Inflammation is the body's defense reaction to injuries such as those caused by mechanical damage, infection or antigenic stimulation. An inflammatory reaction may be expressed pathologically when inflammation is induced by an inappropriate stimulus such as an autoantigen, is expressed in an exaggerated manner, or persists well after the removal of the injurious agents. Such inflammatory reaction may include the production of certain cytokines.
While the etiology of inflammation is poorly understood, considerable information has recently been gained regarding the molecular aspects of inflammation. This research has led to identification of certain cytokines which are believed to figure prominently in the mediation of inflammation. Cytokines are extracellular proteins that modify the behavior of cells, particularly those cells that are in the immediate area of cytokine synthesis and release. Tumor necrosis factors (TNFs) are a class of cytokines produced by numerous cell types, including monocytes and macrophages.
At least two TNFs have been previously described, specifically TNF alpha (TNF-α) and TNF beta (TNF-β or lymphotoxin), and each is active as a trimeric molecule and is believed to initiate cellular signaling by crosslinking receptors; Engelmann et al., J. Biol. Chem., 265:14497-14504 (1990).
Several lines of evidence implicate TNF-α and TNF-β as major inflammatory cytokines. These known TNFs have important physiological effects on a number of different target cells which are involved in inflammatory responses to a variety of stimuli such as infection and injury. The proteins cause both fibroblasts and synovial cells to secrete latent collagenase and prostaglandin E2 and cause osteocyte cells to stimulate bone resorption. These proteins increase the surface adhesive properties of endothelial cells for neutrophils. They also cause endothelial cells to secrete coagulant activity and reduce their ability to lyse clots. In addition they redirect the activity of adipocytes away from the storage of lipids by inhibiting expression of the enzyme lipoprotein lipase. TNFs also cause hepatocytes to synthesize a class of proteins known as “acute phase reactants,” which act on the hypothalamus as pyrogens; Selby et al., Lancet, 1(8583):483 (1988); Starnes, Jr. et al., J. Clin. Invest., 82:1321 (1988); Oliff et al., Cell, 50:555 (1987); and Waage et al., Lancet, 1(8529):355 (1987). Additionally, preclinical results with various predictive animal models of inflammation, including rheumatoid arthritis, have suggested that inhibition of TNF can have a major impact on disease progression and severity; Dayer et al., European Cytokine Network, 5(6):563-571 (1994) and Feldmann et al., Annals Of The New York Academy Of Sciences, 66:272-278 (1995). Moreover, recent preliminary human clinical trials in rheumatoid arthritis with inhibitors of TNF have shown promising results; Rankin et al., British Journal Of Rheumatology, 3(4):4334-4342 (1995); Elliott et al., Lancet, 344:1105-1110 (1995); Tak et al., Arthritis and Rheumatism, 39:1077-1081 (1996); and Paleolog et al., Arthritis and Rheumatism, 39:1082-1091 (1996).
Protein inhibitors of TNF are disclosed in the art. EP 308378 reports that a protein derived from the urine of fever patients has a TNF inhibiting activity. The effect of this protein is presumably due to a competitive mechanism at the level of the receptors. EP 308378 discloses a protein sufficiently pure to be characterized by its N-terminus. The reference, however, does not teach any DNA sequence or a recombinantly-produced TNF inhibitor.
Recombinantly-produced TNF inhibitors have also been taught in the art. For example, EP 393438 and EP 422339 teach the amino acid and nucleic acid sequences of a mature, recombinant human “30 kDa TNF inhibitor” (also known as a p55 receptor and as sTNFR-I) and a mature, recombinant human “40 kDa inhibitor” (also known as a p75 receptor and as sTNFR-II) as well as modified forms thereof, e.g., fragments, functional derivatives and variants. EP 393438 and EP 422339 also disclose methods for isolating the genes responsible for coding the inhibitors, cloning the gene in suitable vectors and cell types, and expressing the gene to produce the inhibitors. Mature recombinant human 30 kDa TNF inhibitor and mature recombinant human 40 kDa TNF inhibitor have been demonstrated to be capable of inhibiting TNF (EP 393438, EP 422339, PCT WO 92/16221 and PCT WO 95/34326).
sTNFR-I and sTNFR-II are members of the nerve growth factor/TNF receptor superfamily of receptors which includes the nerve growth factor receptor (NGF), the B cell antigen CD40, 4-1BB, the rat T-cell antigen MRC OX40, the Fas antigen, and the CD27 and CD30 antigens; Smith et al., Science, 248:1019-1023 (1990). The most conserved feature amongst this group of cell surface receptors is the cysteine-rich extracellular ligand binding domain, which can be divided into four repeating motifs of about forty amino acids and which contains 4-6 cysteine residues at positions which are well conserved; Smith et al., supra.
EP 393438 further teaches a 40 kDa TNF inhibitor •51 and a 40 kDa TNF inhibitor •53, which are truncated versions of the full-length recombinant 40 kDa TNF inhibitor protein wherein 51 or 53 amino acid residues, respectively, at the carboxyl terminus of the mature protein are removed. Accordingly, a skilled artisan would appreciate that the fourth domain of each of the 30 kDa TNF inhibitor and the 40 kDa inhibitor is not necessary for TNF inhibition. In fact, various groups have confirmed this understanding. Domain-deletion derivatives of the 30 kDa and 40 kDa TNF inhibitors have been generated, and those derivatives without the fourth domain retain full TNF binding activity while those derivatives without the first, second or third domain, respectively, do not retain TNF binding activity; Corcoran et al., Eur. J. Biochem., 223:831-840 (1994); Chih-Hsueh et al., The Journal of Biological Chemistry, 270(6):2874-2878 (1995); and Scallon et al., Cytokine, 7(8):759-770 (1995).
PCT WO US97/12244 describes functionally active truncated forms of sTNFR-I and sTNFR-II (referred to as “truncated sTNFR(s)). The truncated sTNFRs are modified forms of sTNFR-I and sTNFR-II which do not contain the fourth domain (amino acid residues Thr127-Asn161 of sTNFR-I and amino acid residues Pro141-Thr179 of sTNFR-II); a portion of the third domain (amino acid residues Asn111-Cys126 of sTNFR-I and amino acid residues Pro123-Lys140 of sTNFR-II); and, optionally, which do not contain a portion of the first domain (amino acid residues Asp1-Cys19 of sTNFR-I and amino acid residues Leu1-Cys32 of sTNFR-II).
PEG-rmet-Hu-sTNF-R (PEGsTNF-R) as described herein is a recombinant form of a functionally active truncated form of sTNFR-I and sTNFR-II which has been PEGylated at the N-terminus with, e.g., a 30 kDa polyethylene glycol molecule. In our preliminary studies with PEGsTNF-R1 it was found that as the PEGsTNF-R1 is concentrated, the viscosity of the solution increases exponentially. Large scale methods traditionally used for concentrating proteins are known to be unsatisfactory when working with such viscous solutions, and the increased viscosity may prevent concentrating the protein to high concentrations without damaging the final product. Because there may be instances in a commercial setting where it will be necessary to have the protein at a higher concentration (e.g., >45 mg/ml) in order to deliver to required therapeutic dose, there is a need to develop formulations which obtain such concentrations and with acceptable low viscosities (e.g., <400 cP) to allow for the use of the various delivery devices necessary for delivery of the therapeutic dose. For example, in order to deliver the required therapeutic dose of a PEGsTNF-R1 formulation wherein the PEGsTNF-R1 concentration is >45 mg/ml, and using a commercially available autoinjector and pre-filled syringe as the delivery device, the formulation should have a viscosity of <400 cP. Above this viscosity, the strong possibility exists for the device or container to fail. The present invention provides for PEGsTNF-R1 formulations having such concentrations and low viscosities, thereby allowing for use of delivery devices which are more convenient and patient-friendly.